Abstract Piezo1 and Piezo2 are mammalian cation-selective mechanosensitive ion channels homologs which open their pore in response to various mechanical stimuli. Mechanotransduction signaling through Piezo channels plays a central role in a bewildering variety of important physiological processes including red blood cell osmotic homeostasis, somatic and visceral mechanosensation, proprioception, blood pressure regulation and development and differentiation of many tissues and organ systems. Several human diseases including xerocytosis and lymphedema have been directly linked to genetic mutations in Piezo channels and many studies further indicate a role of Piezo-mediated signaling in allodynia and hyperalgesia and a possible role of Piezo channels in sleep apnea. The development of drugs capable of selectively activating or inhibiting Piezo channels represent a promising therapeutic opportunity for the treatment of some of these Piezo-related pathologies. To date, Yoda1, a synthetic small molecule agonist capable of selectively activating Piezo1 with micromolar affinity, represents the best small molecule candidate to expand the pharmacome of Piezo channels. Unfortunately, the fundamental mechanisms by which Piezo channel sense mechanical forces and activates in the presence of Yoda1 are still unknown. In this proposal we will address these two unsolved questions using a multidisciplinary approach combining molecular dynamic (MD) stimulations and experimental assays. In our first aim, we will identify rapid, force-induced structural rearrangements in Piezo1 by simulating the channel molecule in a membrane under tension. On another hand, using force-clamp fluorimetry, we will probe local conformational changes using spectroscopic measurements. This will be done by inserting conformational probes into strategic positions of the channel expressed in cells while protein function is being monitored in real-time. This combination of computations and experiments will allow us to capture structural dynamic information that happens in a temporal window spanning several orders of magnitude, from microsecond to minutes. In our second Aim, we will identify how Yoda1 interacts with and activates Piezo1. We have already identified a Yoda1 binding site using a combination of predictive MD simulations and experimental validations. We will characterize structural changes, changes in transition free energy, and modifications of allosteric residue-residue interactions that happen upon Yoda1 binding. This aim will shed light on the mechanism of chemical activation of a Piezo channel and will be invaluable to develop pharmacological agents with clinical value.